Compounds with anti-KS and anti-HIV activity

ABSTRACT

The present invention relates to a compound having anti-KS and anti-HIV pharmaceutical activity which comprises an HCG-like inhibitory protein and fragments or derivatives thereof, said protein and fragments thereof are isolated from a biologically active fraction of APL-HCG, wherein said protein has a molecular weight of about 3,500 or of about 13,000 Dalton, and wherein said protein and fragments thereof are adsorbed polypropylene plastic supports. A pharmaceutical composition for the prevention and/or treatment of Kaposi&#39;s sarcoma (KS) and HIV which comprises an therapeutically effective amount of at least one compound of the present invention in association with a pharmaceutically acceptable carrier. A method for the prevention, treatment and/or reduction of Kaposi&#39;s sarcoma and HIV expression in AIDS patients, which consists in administering the composition to the patient.

[0001] This application is a continuation of PCT/CA98/00731 filed Jul.30, 1998 designating the United States and claiming priority of U.S.provisional Patent Application Ser. No. 60/054,543 filed Aug. 1, 1997.

BACKGROUND OF THE INVENTION

[0002] (a) Field of the Invention

[0003] The invention relates to compounds which exhibit anti-KS andanti-HIV activity, pharmaceutical compositions and method of treatmentthereof.

[0004] (b) Description of Prior Art

[0005] Kaposi's sarcoma (KS) is the most common tumour in AIDS subjectswhich afflicts high mortality (Friedman-Kien A E et al., 1990, J Am AcadDermatol 22:1237-1250). Less aggressive forms can also occur in non-AIDSsubjects of the Mediterranean area and equatorial Africa as well as inrenal transplant patients following treatment with immunosuppressivedrugs (Friedman-Kien A E et al., 1990, J Am Acad Dermatol 22:1237-1250).The pathogenesis and therapy of KS remain enigmatic (Bais C. et al.,1998, Nature 391:86). For unknown reasons, occurrence of KS is higher inmales than in females. For example, in the West, approximately 95% ofAIDS-KS subjects are men. Although, hormonal dependence of KS has beendemonstrated in the case of glucocorticoid and retinoid (Guo W X et al.,1996, Am J Pathol 148: 1999-2008; Guo W X et al., 1995, Am J Pathol 146:727-734; Guo W X et al., 1995, Cancer Res 55: 823-829), sex steroids donot seem to be directly involved in KS pathogenesis. Recently,Lunardi-Iskandar et al. (Lunardi-Iskandar Y et al., 1995, Nature 375:64-68) reported that the placental hormone human chorionic gonadotropin(HCG), displays anti-KS activity and prevents tumours in immunodeficientmice. This preliminary finding could potentially have significanttherapeutic impact as demonstrated in clinical trials (Gill P S et al.,1996, New Engl J Med 335: 1261-1310; Gill P S et al., 1997, J. Natl.Cancer Inst. 89: 1797) and may shed light on basic understanding of thisdisease particularly regarding the sexual dimorphism issue. Though therole of HCG is principally to sustain pregnancy, it is becomingincreasingly apparent that this hormone, possibly along with otheractive molecules, may be responsible for numerous other phenomena. Thelow transmission rate of HIV across the placenta (Prober C G et al.,1991, Ped Infect Dis J 10: 684-695) as well as the low incidence ofKaposi Sarcoma in women including those previously infected with thevirus has led to the suspicion that pregnancy and/or reproductivehormones (such as related LH, see Lunardi-Iskandar Y et al., 1995,Nature 377: 21-22) may be involved in curtailing the propagation of thevirus. Studies by Bourinbaiar (Bourinbaiar A S et al., 1995, ImmunolLett 44: 13-18) indicate that the hormone HCG or its β subunit may havean anti-HIV effect. The action of HCG on gonadal cells is mediated by aG-protein coupled trans-membrane receptor which interacts with thedimeric hormone (α and β complex) with very high affinity andspecificity (review Segaloff D L et al., 1993, Endocr Rev 14: 324-347).In such a system, it is very well known that either one of theindividual α and β subunits have extremely low reactivity towards themembrane bound receptor (Pierce J G et al., 1981, Ann Rev Biochem 50:465-495; Sairam M R, 1983, In: Hormonal Proteins and Peptides. Li C. H.,ed., pages 1-79) but complete activity can be regained by appropriate(1:1) recombination of the two subunits. Lunardi-Iskandar's andBourinbaiar's data (Lunardi-Iskandar Y et al., 1995, Nature 375: 64-68;Bourinbaiar A S et al., 1995, Immunol Lett 44: 13-18) suggest theinvolvement of an “unconventional” mode of action for HCG in KS. Infact, they reported a biological activity for the β HCG, a notion whichcontradicts the generally accepted paradigm that the dimeric form of thehormone is required for triggering hormonal responses in classicaltarget tissues. While stirring a controversy (Lunardi-Iskandar Y et al.,1995, Nature 377: 21-22; Berger P et al., 1995, Nature 377: 21; Rabkin CS et al., 1995, Nature 377: 21-22; Krown S E, 1996, New Engl J Med 335:1309-1310), these findings raise intriguing and potentially novelissues.

[0006] There is reported in Nature Medicine (Vol. 4, No. 7, July 1998)that the anti-KS activity of crude hCG preparations is still a mystery.

[0007] It would be highly desirable to be provided with compounds whichwould exhibit anti-KS and anti-HIV activity.

SUMMARY OF THE INVENTION

[0008] One aim of the present invention is to provide with compoundswhich would exhibit anti-KS and anti-HIV activity.

[0009] In accordance with one preferred embodiment of the presentinvention there is provided a compound having anti-KS and anti-HIVpharmaceutical activity which comprises an HCG-like inhibitory proteinand fragments thereof, the protein and fragments thereof are isolatedfrom a biologically active fraction of APL-HCG, wherein said protein hasa molecular weight of about 3,500 or of about 13,000 Dalton, and whereinsaid protein and fragments thereof are adsorbed to polypropylene plasticsupports, such as tubes or pipette tips among others.

[0010] A preferred polypropylene plastic tube includes those sold bySarstedt (Numbreht, Germany) cat # 57.512 and cat # 68.752.

[0011] In accordance with another preferred embodiment of the presentinvention there is provided purified protein and derivatives andfragments thereof having anti-KS and anti-HIV pharmaceutical activitywhich is a HCG-like inhibitory protein and derivatives and fragmentsthereof which are adsorbed to polypropylene plastic supports, andwherein said protein has an amino acid sequence selected from the groupconsisting of :

[0012]Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg-Glu-Arg-Pro-Ile-Asn*-Ala-Thr-Leu-Ala-Val-Glu-LysSEQ ID NO:1; and

[0013] Ala-Pro-Asp-Val-Gln-Asp-Lys-Phe-Thr-Arg-Gln-Ile-Met-Ala-Thr SEQID NO:2.

[0014] The purified protein of the present invention is referred to asHIP or HCG-like Inhibitory Protein.

[0015] In other embodiments, the derivatives contain one or more D-aminoacids or non-natural amino acids.

[0016] In accordance with another preferred embodiment of the presentinvention there is provided a pharmaceutical composition for theprevention and/or treatment of Kaposi's sarcoma (KS) and/or HIV whichcomprises a therapeutically effective amount of at least one compound ofthe present invention in association with a pharmaceutically acceptablecarrier.

[0017] In accordance with another preferred embodiment of the presentinvention there is provided a pharmaceutical composition for theprevention and/or treatment of Kaposi's sarcoma (KS) and/or HIV whichcomprises a therapeutically effective amount of at least one protein ofthe present invention in association with a pharmaceutically acceptablecarrier.

[0018] In other embodiments, the pharmaceutical composition isformulated as a controlled release formulation.

[0019] In accordance with another preferred embodiment of the presentinvention there is provided a pharmaceutical composition for theprevention and/or treatment of Kaposi's sarcoma (KS) and/or HIV whichcomprises a therapeutically effective amount of a derivative of aprotein having anti-KS and anti-HIV pharmaceutical activity which is aHCG-like inhibitory protein which is adsorbed to polypropylene plasticsupports, and wherein said protein has an amino acid sequence selectedfrom the group consisting of :

[0020]Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg-Glu-Arg-Pro-Ile-Asn*-Ala-Thr-Leu-Ala-Val-Glu-LysSEQ ID NO:1; and

[0021] Ala-Pro-Asp-Val-Gln-Asp-Lys-Phe-Thr-Arg-Gln-Ile-Met-Ala-Thr SEQID NO:2

[0022] in association with a pharmaceutically acceptable carrier.

[0023] In accordance with another preferred embodiment of the presentinvention there is provided a method for the prevention, treatmentand/or reduction of Kaposi's sarcoma and/or HIV expression in AIDSpatients, which comprises administering to said patient atherapeutically effective amount of a compound of the present invention.

[0024] In accordance with another preferred embodiment of the presentinvention there is provided a method for the prevention, treatmentand/or reduction of Kaposi's sarcoma and/or HIV expression in AIDSpatients, which comprises administering to said patient atherapeutically effective amount of a protein of the present invention.

[0025] In accordance with another preferred embodiment of the presentinvention there is provided a method for the prevention, treatmentand/or reduction of Kaposi's sarcoma and/or HIV expression in AIDSpatients, which comprises administering to said patient atherapeutically effective amount of a pharmaceutical composition of thepresent invention.

[0026] In accordance with another preferred embodiment of the presentinvention there is provided a method to purify the compound or proteinof the present invention, which comprises the steps of:

[0027] a) subjecting a biologically active fraction of APL-HCG orurinary extract containing said compound or protein to a polypropyleneplastic support for a time sufficient for adsorption of said compound orprotein to occur; and

[0028] b) washing the support and releasing the adsorbed compound orprotein therefrom.

[0029] In accordance with another preferred embodiment of the presentinvention there is provided a method of evaluating inhibitory activityof anti-KS and anti-HIV compound, which comprises by measuring AP1 geneactivity.

[0030] In other embodiments, measuring of said AP1 gene activity iseffected by measuring binding to DNA response element.

[0031] For the purpose of the present invention the following terms aredefined below.

[0032] “HIP” : HCG-like Inhibitory Protein;

[0033] “HPLC” : high-pressure liquid chromatography; and

[0034] “APL” : commercial trade name of the clinical-grade HCG sold byWyeth-Ayerst, cat. # DIN 02168936.

[0035] The expression “derivatives and fragments thereof” is intended tomean any derivatives and fragments of the protein of the presentinvention which exhibit anti-KS and anti-HIV pharmaceutical activityeffective for the prevention, treatment and/or reduction of Kaposi'ssarcoma in AIDS patients. The derivatives may include one or moreD-amino acids or non-natural amino acids. The derivatives and fragmentsare functional and substantially exhibit the biological activity of theprotein of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1 illustrates the effect of HCG from different commercialsources on KS-Y1 cell proliferation;

[0037]FIG. 2 illustrates the fractionation and activity profile ofAPL-HCG;

[0038]FIG. 3 illustrates the time-course effect of APL-HCG on inhibitionof AP-1 binding in KSY-1 cells;

[0039]FIG. 4 illustrates the purification of the HIP using reversedphase-HPLC;

[0040]FIG. 5 illustrates the bioassay of the collected fractionsfollowing HPLC separation;

[0041]FIG. 6 illustrates the analysis of fraction D by massspectrometry; and

[0042]FIG. 7 illustrates the analysis of fraction A+B+C+E by massspectrometry;

[0043]FIG. 8 illustrates the analysis of another low molecular weightfraction by mass spectrometry;

[0044]FIG. 9 illustrates the effect of HIP on HIV expression; and

[0045]FIGS. 10A and 10B illustrate potential partial sequences of thepurified HIP protein of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0046] Kaposi's sarcoma (KS), a sexually dimorphic disease inflictinghigh mortality in AIDS, remains at present without effective treatment.A recent report (Lunardi-Iskandar Y et al., 1995, Nature 375: 64-68)showed that the placental glycoprotein hormone, human chorionicgonadotropin (HCG), and surprisingly its β subunit, inhibittumorigenicity and metastasis of Kaposi's sarcoma cells in micexenografts. The anti-KS efficacy of a commercial HCG was subsequentlydemonstrated in clinical trials (Gill P S et al., 1996, New Engl J Med335: 1261-1310; Gill P S et al., 1997, J. Natl. Cancer Inst. 89: 1797).In addition, earlier studies by Bourinbaiar (Bourinbaiar, A.S. et al.,1992, FEBS. Lett. 309: 82-84; Bourinbaiar A S et al., 1995, Immunol Lett44: 13-18) and by Gallo's group (Lunardi-Iskandar Y et al., 1998, NatureMedicine 4:428-434) indicate that the β subunit of HCG (or peptidesderived thereof) have anti-HIV effects.

[0047] The Applicants have been working for several years on thecellular and molecular aspects of KS regulation by hormones. Recentstudies in the Applicants' laboratory confirm that commercial HCGpreparations (known to be about 25% pure) display significant inhibitoryaction in a dose-dependent manner. However, pure and biologically activeHCG has no effect on Kaposi's sarcoma growth in culture suggesting thata contaminant (or degradation product) may be the active agent.

[0048] The Applicants have subfractionated commercial HCG preparationsbased on molecular size and each fraction was tested with respect toinhibition of KS cell growth, HCG radioreceptor binding andsteroidogenic bioactivity. The Applicants' results demonstrate that theanti-KS activity resides among low molecular weight components, and notin bona fide (macromolecular) HCG. Interestingly, the Applicants haveidentified a transcription factor which may be the target for regulationby the anti-KS components. The Applicants have concluded that, as yetunidentified molecules, present in the commercial HCG preparations, areresponsible for the growth inhibitory effects wrongfully attributed toHCG.

[0049] Surprisingly, and in accordance with the present invention, thereis provided the identification of a purified HIP protein having anti-KSand anti-HIV pharmaceutical activity. This protein is an HCG-likeinhibitory protein and is adsorbed to polypropylene plastic supports,and has an amino acid sequence selected from the group consisting of :

[0050]Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg-Glu-Arg-Pro-Ile-Asn*-Ala-Thr-Leu-Ala-Val-Glu-LysSEQ ID NO:1; and

[0051] Ala-Pro-Asp-Val-Gln-Asp-Lys-Phe-Thr-Arg-Gln-Ile-Met-Ala-Thr SEQID NO:2.

[0052] Sources of HCG

[0053] Two commercial HCG samples were tested. The first one, under thetrade name of APL, was provided by Wyeth-Ayerst, Montréal (Lot # C84662Awas generously donated and cat. # DIN-02168936 was purchased), it shouldbe emphasized that APL was used in the earlier studies (Lunardi-IskandarY et al., 1995, Nature 375: 64-68; Gill P S et al., 1996, New Engl J Med335: 1261-1310). Two samples were also purchased from Sigma, St-Louis,Mo. (lot # 26H 1040). Pure HCG dimer as well as α-HCG and β-HCG wereobtained from NIDDK (Bethesda, Md.). Recombinant HCG was obtained fromOrganon, Oss, the Netherlands. All HCG samples, previously storedlyophilised, were dissolved in PBS and frozen as aliquots.

[0054] Assessment of Cell Proliferation

[0055] The KS-Y1 (Lunardi-Iskandar Y et al., 1995, Nature 375: 64-68)was isolated from an HIV-patient while. the subline designated N-1506(Lunardi-Iskandar Y et al., 1995, Nature 375: 64-68) of the originalKS-SLK cell line originated from an immunosupressed subject (Herndier BG et al., 1994, AIDS 8: 575-581). These cell lines were provided by Dr.Lunardi-Iskandar (N. I. H., Bethesda). The KS cells were passaged andthe culture medium was changed every other day in presence or in absenceof any of the HCG samples mentioned above for the indicated periodsranging from 24-96 hrs. ³H-thymidine incorporation was measured asdescribed (Guo W X et al.,. 1996, Am J Pathol 148: 1999-2008; Guo W X etal., 1995, Am J Pathol 146: 727-734) In most experiments, data arereported as means ±SEM of quadruplet determinations. Statisticalanalysis was determined by student t-test.

[0056] Fractionation of APL on SEPHADEX™ G-100

[0057] Three vials of APL (10 000 IU/vial) were pooled for fractionationby dissolving in 1.5 ml of 0.05 M NH₄ HCO₃. The clear solution wasloaded on a column of SEPHADEX™ G-100 (Pharmacia, Baie d'Urfé, Qc)(1.5×90 cm) equilibrated in the same solvent. Fractions of 1.7 ml werecollected and pooled into seven fractions (see FIG. 3). A small portionof each was saved for estimating HCG equivalent activity and theremainder was lyophilized.

[0058] HCG Receptor Binding Activity

[0059] A convenient test for HCG, a hormone which efficiently binds tothe LH receptor, is to perform radioreceptor assays using membranepreparations of adult pig testes as described in detail (Sairam M R,1983, In: Hormonal Proteins and Peptides. Li C. H., ed., pages 1-79;Manjunath P et al., 1982, J Biol Chem 257: 7109-7115). Standard (CR-125HCG from NICHD, Bethesda) or test samples were tested for ¹²⁵I-HCGbinding as described (Sairam M R, 1983, In: Hormonal Proteins andPeptides. Li C. H., ed., pages 1-79). The total binding activity in eachof the seven fractions was calculated and expressed as, ug HCGequivalent per fraction.

[0060] Steroidogenic Activity

[0061] HCG is a highly potent steroidogenic hormone, therefore onereliable bioassay consists of incubating mouse Leydig tumour cells(MA-10, originally obtained from Dr. M. Ascoli, Iowa) with the testmaterial as described (Sairam M R, 1983, In: Hormonal Proteins andPeptides. Li C. H., ed., pages 1-79). Progesterone in the medium wasestimated by radioimmunoassay (Sairam M R, 1983, In: Hormonal Proteinsand Peptides. Li C. H., ed., pages 1-79).

[0062] Electrophoretic Mobility Gel Shift Assay (EMSA)

[0063] Nuclear extracts were prepared from KSY-1 cell cultures accordingto the original procedure of Smeal (Smeal T et al., 1989,. GenesDevelop. 3:2091-2100). Binding reactions for AP-1 sites (TRE, TPAResponse Element) were carried out as described (Smeal T et al., 1989,.Genes Develop. 3:2091-2100, and reviewed in Saatcioglu F et al., 1994,Semin. Cancer Biol. 5:347-359). Synthetic collagenase TREoligonucleotide probe of the sequence 5′-GGATCCGATGAGTCAGCCA-3′ was endlabelled with ³²P-ATP and EMSA performed as described (Smeal T et al.,1989,. Genes Develop. 3:2091-2100). Specificity was ascertained by using100 molar excess of unlabelled TRE. The signal was quantified byphosphorimager analysis using the software by Molecular Dynamics(Sunnyvale, Calif.).

[0064] Pure HCG has No Inhibitory Activity in KS Cells (FIG. 1)

[0065] Initial experiments were designed to confirm the inhibitoryaction of HCG. The effects on the two different KS cell lines werecompared. In cells pre-treated with a commercial HCG preparation (Sigmaor APL) an inhibitory effect was elicited (p<0.05) in all KS cell lines.In preliminary experiments a dose-dependent inhibition of cell growthwas noted.

[0066] The two commercial HCG products (APL and Sigma) were tested, andnear identical inhibition was obtained (FIG. 1), right-hand two bars).However, some HCG shipments were more potent than others.

[0067] Samples were used at an equivalent concentration of 50 U/ml (FIG.1). Note that upon treatment with Sigma-HCG (S) or Ayerst-HCG (APL), KScell growth was significantly reduced as compared with thevehicle-treated cells (C). In contrast, no inhibitory effect was notedusing preparations of highly purified HCG. Legend: 1=dimeric HCG; 2=αHCG; 3=β HCG; 4=unrelated human urinary protein pool; *p<0.05.

[0068] Next, the anti-KS activity of a well characterized, pure dimericHCG, pure α or β subunits and recombinant HCG was verified. Neither oneof these pure HCG's inhibited KS growth (FIG. 1, #1-3). The biologicalactivity of these compounds was examined by induction of steroidogenesisin cultured Leydig cells. As expected, either recombinant or pure HCGelicited the classic biological responses, while neither α nor β HCGdisplayed any steroidogenic action.

[0069] Molecular Sieving of Crude HCG (FIG. 2)

[0070] Generally, the pregnancy hormone ampouled into vials for clinicaluse is only about 25% pure for HCG as evaluated by biological activityand biochemical analyses (Manjunath P et al., 1982, J Biol Chem 257:7109-7115). The commercial HCG (APL) was sorted into 7 distinctfractions using SEPHADEX™ chromatography (FIG. 2).

[0071] The contents of 3 vials of clinical grade APL (10,000 IU each)were dissolved in 0.05 M NH₄ HCO₃ and subjected to molecular sieving ona column of SEPHADEX™ G-100 (1.5×90 cm). The eluted protein/peptidefractions monitored at A230 nm (panel E) were separated into seven poolsidentified as fraction pools # 1-7 on the X-axis. A total of 120 tubes(1.75 ml/tube) were collected. Lyophilized material in each pool wasreconstituted in KS culture medium (without serum), and evaluated forcell proliferation (panel A). HCG receptor binding in pig testicularmembranes (panel B) and steroidogenic activity in MA-10 cells (panel C)were determined. Panel D: bar graphs show quantitative densitometricscanning of AP-1 binding and insert shows the actual EMSA protein-DNAcomplexes of fractions (fr) 2,4 and 7; nd=not determined. KSY-1 cellswere treated with the indicated reagents at an equivalent concentration100 U/ml for 4 days. Note clear segregation of HCG hormone activity ongonadal cells (pool 2) and inhibitory action on KS cells (pool 7)*p<0.05.

[0072] Over 85% HCG receptor binding activity (FIG. 2B) was recovered inthe first two pooled fractions where high molecular weight proteins ofthe size of pure HCG would emerge. The Ve/Vo ratio of the early majorfraction (pool #2) corresponded to bona fide HCG. These fractions mayalso contain the hormone subunits (α/β) or their degraded products inaddition to other unidentified materials present in the crude extract.Fraction #7 consists, as shown in previous studies (Sairam M R, 1983,In: Hormonal Proteins and Peptides. Li C. H., ed., pages 1-79), ofrelatively small peptides along with other agents present in the APLformulation. Either the steroidogenic or the binding activity that ischaracteristic of HCG (but not its subunits) was highest in the 2 ndfraction (FIGS. 2B and C). These results are consistent with receptorbinding assays in which only the dimeric (α-β combined) HCG but not theindividual subunits or their cleaved products are biologically active(Sairam M R, 1983, In: Hormonal Proteins and Peptides. Li C. H., ed.,pages 1-79; Manjunath P et al., 1982, J Biol Chem 257: 7109-7115). Onthe contrary, only fraction #7 contained KS inhibitory activity.

[0073] Down-Regulation of AP-1 Binding by HCG Components (FIG. 3)

[0074] Activating protein-1 (AP-1) is a transcriptional activator whichis induced by 12-O-tetradecanyl phorbol-13-acetate (TPA) tumor promoter,several growth factors and various extracellular stimuli (reviewed inSaatcioglu F et al., 1994, Semin. Cancer Biol. 5:347-359). AP-1 consistsof proteins of jun and fos families which associate to formhomo-(jun/jun) or heterodimers (jun/fos) and recognize a consensussequence 5′-TGA G/C TCA-3′ known as TPA Response Element (TRE) presenton AP-1 regulated genes. AP-1 complexes are considered to play importantroles in several signal transduction pathways such as growthstimulation, differentiation, neuronal excitation and transformation(Saatcioglu F et al., 1994, Semin. Cancer Biol. 5:347-359). APL-HCG andcomponents in fraction 7 significantly inhibited AP-1 binding to TRE inKSY-1 cells (FIG. 2D). APL-HCG inhibited AP-1 binding by 1.5, 3 and 2fold respectively after 3, 6 and 12 hours of treatment (FIG. 3).

[0075] Cells were incubated-with 50 IU/ml APL-HCG (+) or with vehicle(−) for the indicated time periods (FIG. 3). Nuclear extracts wereprepared and EMSA was performed. Results shown are representative ofthree experiments. Arrow-head points to free probe. 100 ex.=100 foldexcess unlabelled probe. Top shows the actual gel shifts while bottompanel provides quantitative phosphorimager measurement of the major band(arrow); * denotes p<0.05 as compared to vehicle-treated.

[0076] A dose-response was also observed with near maximal effect notedat approximately 100 IU/ml. Therefore, repression of AP-1 may be animportant pathway by which inhibition of KSY-1 cells occurs.

[0077] Purification of the HIP Using Reversed Phase-HPLC (FIG. 4)

[0078] APL was purchased from Wyeth-Ayerst Cat. # DIN 02168936 andshipped in an insulated box packed with refrigirant. Upon receipt, APLwas stored at 4° C. One APL vial (wich contained the dried product) lot# JA(L)3YYF-AB was reconstituted with one (1) ml of the solvent soldwith the APL ampoule at room temperature and processed for HPLC withinone hour. The powder was readily dissolved resulting in a homogeneous“solution”. This “solution” was injected into a Waters™ HPLC apparatusfitted with a 7.8×300 mm C-18™ column. Elution from the column was doneusing an increasing linear isocratic gradient of acetonitrile in watercontaining 0.1% trifluoroacetic acid. The gradient was increased from 5%to 75% acetonitrile.The absorbancy was monitored at 220 wavelengthduring the elution and fractions were collected manually in siliconizedpolypropylene tubes. When regular (i.e. non-siliconized) tubes were usedit was later found that biological activity was lost. After collection,the fractions were immediately placed in a Savant™ Speed-vac aparatus inorder to dry the samples. The gradient is drawn on FIG. 4; theright-side or Y axis shows the % acetetonile (% B; B: 80% acetonitrilein water containing 0.1% trifluoroacetic acid) and the X axis indicatestime, in minutes. The absorbency at 220 nm was recorded and recorded onthe Y axis. The two peaks (D & E) indicated by arrows were subsequentlyfound (see FIG. 5 below) to contain the KS inhibitory activity.

[0079] Bioassay of the Collected Fractions Following HPLC Separation(FIG. 5)

[0080] The fractions (peaks) indicated by arrows on FIG. 4. werelyophilized and each was reconstituted in one (1) ml of RPMI culturemedium (without serum) and tested for biological activity using theKS-Y1 cells. Since the original material was supplied as 10 000 IU ofHCG, by analogy, it was assumed arbitrarily that one of the fractionsshould contain arbitrarily 10,000 IU of anti-KS activity. With such anassumption, the doses were evaluated throughout the present application.The biological activity was tested in absence (0) or presence ofdifferent doses (10, 100 & 200 IU/ml) . The fraction indicated as “mix”represents one pool made by mixing equivalent amounts of fractions A-E.It can be seen from FIG. 5 that fractions D, E and “Mix” display aninhibitory activity.

[0081] Analysis of the Active Fractions (HIP) by MALDI-TOF MassSpectrometry (FIGS. 6 and 7)

[0082] Briefly, an aliquot of each sample was embedded in a lowmolecular weight UV-absorbing matrix ((α-cyano-4-hydroxycinnamic acid)to enhance sample ionization and then subjected to MALDI-TOF (MatrixAssisted Laser Desorption Ionization Time of Flight) mass spectrometryon a Voyager-Delayed Extraction system (Perseptive Biosystem,Framingham, Mass.).

[0083] One major peak can be observed containing moieties atapproximately 13000 Dalton (FIG. 6). For comparison purposes, thespectrometric analysis of fractions A-C and E are also shown (FIG. 7),note that the 13000 Dalton species is found in both fractions D and E(FIG. 6).

[0084] Effect of HIP on HIV Expression (FIG. 9)

[0085] The low molecular weight fraction #7 shown earlier to inhibit KScell proliferation (Kachra et al., 1997, Endocrinology, 138:4038-4041),was tested for its anti-HIV activity. Primary cultured human lymphocyteswere infected with the virus HIV-IIIB as described (Tremblay et al.,1994, Embo. J. 13:774). Immediately following infection, cells weretreated with the test material (HCG fractions or recombinant HCG) dailyfor 10 days at the indicated dose ranging from 1 to 250 IU equivalent.Subsequently, cells were lysed and assayed for the expression of the HIVviral protein p24 as described (Tremblay et al., 1994, Embo. J. 13:774).It can be noted that p24 expression is markedly reduced upon treatmentwith high doses of the fraction containing HIP while recombinant HCGdisplays no significant affects (FIG. 9).

[0086] Sequencing of the Proteins Contained in the HIP Fractions (FIGS10A and 10B)

[0087] Following the HPLC separation, the fractions were tested forbiological activity as described above (for FIGS. 4 and 5). Twofractions which contains highest bioactivity were processed for proteinsequencing using an automatic sequencing apparatus (Applied Biosystemgas phase sequencer model 470 updated to 475). An internal standard wasused consisting of pTH-Nor-Leu. The initial yield efficiency wasapproximately 50±20 pmoles. After 15 cycle runs, data generated wasexamined using customary protein analysis. The deduced amino acidsequences were compared with published databases of the GenBank™. It wasfound that the two sequences contained significant homology with the αand β-subunits of hCG.

[0088] Conclusion

[0089] The present results provide evidence for the existence of apotentially important compound which inhibits the growth of KS possiblythrough signalling by the AP-1 pathway. Although the sequencing of thepurified active molecule is currently in progress, it is evident that itis neither HCG nor any of its classically unknown subunits.

[0090] Judging from its gel permeation chromatographic elution, its sizeis relatively small and probably less than 10,000-14,000. To obtainfurther resolution, the technique of HPLC was employed resulting in aseparation of protein species into discrete and distinct peaks. Specificindividual peaks were found to contain the anti-KS activity. To obtainfurther data, individual peaks were analyzed by polyacrylamide gelelectrophoresis followed by silver staining. In instances where proteinswas visualized, a “fuzzy” band was observed, indicating that theproteins comprise closely related species.

[0091] At this time, one can only speculate as to whether it is derivedfrom HCG as a cleavage peptide. Indeed, it is known that glycoproteinhormones are metabolized to smaller polypeptides (Sairam M R, 1983, In:Hormonal Proteins and Peptides. Li C. H., ed., pages 1-79). The putativecleavage (or related product) could elicit its action via a modified HCGreceptor. In fact, the HCG receptor gene is known to be expressed asalternatively spliced variant transcripts (Segaloff D L et, al., 1993,Endocr Rev 14: 324-347) in a developmentally regulated manner raisingthe possibility that the putative product could mediate differentaspects of hormone action. Such a hypothesis is further strengthened byparallel experiments showing that KS tissues and cell lines expresssignificant levels of HCG receptors whose size and intracellulardistribution are different from classical targets cells (Cao H., SairamM. R. and Antakly T., Abstract # 1543, Annual meeting, AmericanAssociation for Cancer Research, 1996). Alternatively, the activesubstance could be a degradation product of the β-HCG subunit (such asbut not limited to β-core) which is homologous in three-dimensionalstructure to several growth factors (Lapthorn A J et al., 1994, Nature369: 455-461). Since the initiation and proliferation of KS cells islargely growth factor-dependent, it is possible that β-core fragmentsact as antagonists for growth factor receptors (reviewed in Guo W X etal., 1996, Am J Pathol 148: 1999-2008).

[0092] The partial sequences obtained in accordance with the presentinvention support the view that HIP proteins may be derived from hCGeither as: 1) alternate expression of α or β-subunit; or 2) enzymaticprocessing of the hCG subunits.

[0093] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1 4 1 20 PRT Artificial Sequence anti-KS and anti-HIV compound 1 Ser LysGlu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu 1 5 10 15 AlaVal Glu Lys 20 2 13 PRT Artificial Sequence anti-KS and anti-HIVcompound 2 Ala Pro Asp Val Gln Asp Lys Phe Thr Arg Gln Ile Met 1 5 10 315 PRT Artificial Sequence anti-KS and anti-HIV compound 3 Ala Pro AspVal Gln Asp Lys Phe Thr Arg Gln Xaa Met Xaa Xaa 1 5 10 15 4 20 PRTArtificial Sequence anti-KS and anti-HIV compound 4 Ser Lys Glu Pro LeuArg Pro Arg Xaa Arg Pro Ile Asn Ala Thr Leu 1 5 10 15 Ala Val Glu Lys 20

What is claimed is:
 1. A plastic-adsorbing compound having anti-KS andanti-HIV pharmaceutical activity which comprises at least onebiologically active compound isolated from a biologically activefraction of APL-HCG.
 2. A plastic-adsorbing compound of claim 1, whereinsaid at least one biologically active compound has a molecular weight ofless than about 14,000 Dalton.
 3. The plastic-adsorbing compound ofclaim 1, wherein said at least one biologically active compound has anamino acid sequence selected from the group consisting of:Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg-Glu-Arg-Pro-Ile-Asn*-Ala-Tbr-Leu-Ala-Val-Glu-LysSEQ ID NO:1; andAla-Pro-Asp-Val-Gln-Asp-Lys-Phe-Thr-Arg-Gln-Ile-Met-Ala-Thr SEQ ID NO:2;and biologically active derivatives and fragments thereof.
 4. Theplastic-adsorbing compound of claim 3, wherein said derivatives containone or more D-amino acids or non-natural amino-acids.
 5. Apharmaceutical composition for the prevention and/or treatment ofKaposi's sarcoma (KS) and/or HIV which comprises a therapeuticallyeffective amount of at least one compound of claim 1, in associationwith a pharmaceutically acceptable carrier.
 6. A pharmaceuticalcomposition for the prevention and/or treatment of Kaposi's sarcoma (KS)and/or HIV which comprises a therapeutically effective amount of atleast one compound of claim 3, in association with a pharmaceuticallyacceptable carrier.
 7. The pharmaceutical composition of claim 5, whichis formulated as a controlled release formulation.
 8. A method for theprevention, treatment and/or reduction of Kaposi's sarcoma and/or HIVexpression in AIDS patients, which comprises administering to saidpatients a therapeutically effective amount of a compound of claim
 1. 9.A method for the prevention, treatment and/or reduction of Kaposi'ssarcoma and/or HIV expression in AIDS patients, which comprisesadministering to said patient a therapeutically effective amount of apharmaceutical composition of claim5.
 10. A method to purify thecompound of claim 1, which comprises the steps of: a) subjecting abiologically active fraction of APL-HCG or urinary extract comtainingsaid compound or protein to a polypropylene plastic support for a timesufficient for adsorption of said compound or protein to occur; and b)washing the support and releasing the adsorbed compound or proteintherefrom.
 11. A method of evaluating biological anti-KS and anti-HIVactivity of a polypropylene-adsorbing compound of claim 1, whichcomprises measuring AP1 gene activity.
 12. The method of claim 11,wherein measuring of said AP1 gene activity is effected by measuringbinding to DNA response element.